HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 43, 41749-41755, October 24, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/43/41749    most recent M306397200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Malygin, E. G. Articles by Hattman, S. Search for Related Content PubMed PubMed Citation Articles by Malygin, E. G. Articles by Hattman, S. Social Bookmarking                      What's this?

Bacteriophage T4Dam (DNA-(Adenine-N⁶)-methyltransferase)

EVIDENCE FOR TWO DISTINCT STAGES OF METHYLATION UNDER SINGLE TURNOVER CONDITIONS^*

Ernst G. Malygin, William M. Lindstrom, Jr., Victor V. Zinoviev, Alexey A. Evdokimov, Samuel L. Schlagman¶, Norbert O. Reich, and Stanley Hattman¶||

From the Institute of Molecular Biology, State Research Center of Virology and Biotechnology Vector, Koltsovo 630559, Novosibirsk Region, Russia, the Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93103, and the ^¶Department of Biology, University of Rochester, Rochester, New York 14627-0211

We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N⁶)-methyltransferase)-mediated methyl group transfer from S-adenosyl-L-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5'-GATC/5'-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA « 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-L-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages. First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (k_meth1 = 0.21 s^–1); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination. In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (k_meth2 = 0.023 s^–1). We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. The hemimethylated duplex 5'-GATC/5'-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions. The first stage (k_meth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation. The slower second stage (k_meth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain. Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions. When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced.

Received for publication, June 17, 2003 , and in revised form, July 30, 2003.

^* This work was supported by the Russian Foundation for Basic Research (Project No. 01-04-49869), U. S. Public Health Service Grant R03 TW05755 from the Fogarty International Center, National Science Foundation Grant MCB-9983125 (to N. O. R.), and U. S. Public Health Service Grants GM463333 (to N. O. R.) and GM29227 (to S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 585-275-8046; Fax: 585-275-2070; E-mail: modDNA{at}mail.rochester.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				L.-T. Wu, S.-Y. Chang, M.-R. Yen, T.-C. Yang, and Y.-H. Tseng Characterization of Extended-Host-Range Pseudo-T-Even Bacteriophage Kpp95 Isolated on Klebsiella pneumoniae Appl. Envir. Microbiol., April 15, 2007; 73(8): 2532 - 2540. [Abstract] [Full Text] [PDF]

				E. G. Malygin, B. Sclavi, V. V. Zinoviev, A. A. Evdokimov, S. Hattman, and M. Buckle Bacteriophage T4Dam DNA-(Adenine-N6)-methyltransferase: COMPARISON OF PRE-STEADY STATE AND SINGLE TURNOVER METHYLATION OF 40-MER DUPLEXES CONTAINING TWO (UN)MODIFIED TARGET SITES J. Biol. Chem., November 26, 2004; 279(48): 50012 - 50018. [Abstract] [Full Text] [PDF]